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Volume 84 (1978)
- Issue 6 Pages 1323-
- Issue 5 Pages 1021-
- Issue 4 Pages 737-
- Issue 3 Pages 499-
- Issue 2 Pages 245-
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Volume 84, Issue 3
Displaying 1-28 of 28 articles from this issue
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Hiroyuki HASEGAWA, Nobuo NAKANISHI, Miki AKINO1978 Volume 84 Issue 3 Pages 499-506
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe oxidation of tetrahydropterin with ferri-cytochrome c was studied using a tetrahydropteringenerating system composed of dihydropteridine reductase [EC 1. 6. 99. 7] and NADH. Under aerobic conditions, 1.5 to 1.8 mol of cytochrome c was reduced per mol of NADH, whereas 2 mol of cytochrome c was reduced under anaerobic conditions. When superoxide dismutase [EC 1. 15. 1. 1] was added to the system under aerobic conditions, only 1 mol of cytochrome c was reduced per mol of NADH, while the pterin oxidation was scarcely affected. Based on these results, we propose that the oxidation of tetrahydropterin to quinonoid dihydropterin proceeds via two steps: tetrahydropterin is first oxidized by ferri-cytochrome c to give a pterin intermediate, which has lost one electron, then in turn this reduces O2 to form O2-View full abstractDownload PDF (622K) -
Eisuke NISHIDA1978 Volume 84 Issue 3 Pages 507-512
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa ions inhibit microtubule (MT) assembly and induce disassembly of the reconstituted MT. This Ca sensitivity of the MT assembly system varies markedly with the assembly medium. The half-maximal inhibitions of MT reassembly from crude extract (CE) of porcine brain and from purified microtubular proteins (PMP) occurred at free Ca2+ concentrations of 10μM and 700μM, respectively, in the presence of a low Mg concentration (0.5mm) and at low ionic strength (10mm MES and 50mm KCl) (Nishida, E. & Sakai, H. (1977) J. Biochem. 82, 303-306). Increase in the Mg concentration from 0.5 mm to 5 mm decreased the Ca2+ concentrations required for the half-maximal inhibition of CE and PMP from 10 to 2μM and from 700 to about 50μM, respectively. Upon increasing the KCl concentration from 50 to 200mm, the Ca2+ concentration for the half-maximal inhibition of PMP decreased to 10μm from 700μm. Concentrations of KCl higher than 200 mm inhibited MT assembly by suppressing the initiation process. However, the addition of outer fiber fragments permitted growth of MT. This MT elongation was also inhibited by 10μM Ca2+ Moreover, the MT growth induced by mixing purified tubulin dimers with outer fiber fragments showed the same Ca sensitivity as MT assembly from PMP.
RNA decreased the initial rate of MT assembly from PMP, as reported by others. Addition of Ca2+ caused the initial rate to increase, although the final extent of MT assembly was suppressed. However, the Ca sensitivity as determined by the plateau level of the viscosity increase was not significantly altered in the presence of RNA.
High concentrations of Na glutamate as well as glycerol augmented and stabilized the assembled MTs and lowered the Ca sensitivity.View full abstractDownload PDF (428K) -
Hidenori HAYASHI, Tetsuya SUGA1978 Volume 84 Issue 3 Pages 513-520
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSome characteristics of peroxisomes in the slime mold, Dictyostelium discoideum were studied biochemically. The slime mold contained only two peroxisomal enzymes, urate oxidase and catalase. Both activities were concentrated highest in the light mitochondrial fraction, while the highest activity of acid phosphatase as a marker of lysosomes was found in the heavy mitochondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was 1.21-1.22g/ml, and that of lysosomes was approximately 1.21g/ml. When the light mitochondrial fraction was treated with deoxycholate, major activities of urate oxidase, catalase, and acid phosphatase were solubilized. With development of the slime mold, both activities of urate oxidase and catalse increased at the stationary stage (4-6h after) to a great extent and also slightly at the aggregation stage (10-12h), thereafter they decreased gradually. Acid phosphatase remained with moderately high activity till the culmination stage (18-20h), and then disappeared rapidly. It was found that enhanced activities of peroxisomal enzymes at the stationary stage represented an increase in the particle fraction and supernatant. On the other hand, activities of acid phosphatase at the same stage increased two-fold only in the supernatant, while the activity in the particle fraction was reduced to one-half.View full abstractDownload PDF (522K) -
Akira OMORI, Mariko ISHIZUKA-KOBAYASHI, Tsuneko UCHIDA1978 Volume 84 Issue 3 Pages 521-529
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Two main DNases were found in the dried liver extract of a snail, Achatina fulica. They were purified by the phosphocellulose batch method and by phosphocellulose column chromatography. The enzyme eluted earlier from the phosphocellulose column was designated as Achatina DNase-1 and the other as Achatina DNase-2. DNase-1 was purified further by QAE-Sephadex A-25 column chromatography (twice) just before use because of the instability of the purified enzyme. By these procedures, DNase-1 and 2 were purified 200- and 130-fold, respectively.
2. Divalent or monovalent cations had no marked effect on either enzyme. They showed pH optima of 4.8 (DNase-1) and 5.2 (DNase-2). Ionic strength was found to be critical for the maximal activity. The isoelectric points of DNase-1 and 2 were both 6.9. On heating at 70-75°C for 5 min, each enzymic activity fell to half of the initial value.
3. The enzyme preparations degraded native DNA 1.5-2.5 times faster than heat-denatured DNA. They both degraded heat-denatured DNA endonucleolytically, to give oligonucleotides with 3'-phosphates.
4. The 3'-phosphoryl and 5'-hydroxy termini of the resulting oligonucleotides were analyzed. DNase-1 possessed marked specificity for dThd at 3'-termini and dAdo at 5'-termini in the early stages of degradation, but only for dAdo at 5'-termini in the later stages. DNase-2 showed some preference for purine nucleotides at both 3'- and 5'-termini in the later stages of degradation.View full abstractDownload PDF (638K) -
Mapping the Aromatic Binding SiteYuji KARASAKI, Motonori OHNO1978 Volume 84 Issue 3 Pages 531-538
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetic specificities of BPN' and Carlsberg subtilisins [EC 3. 4. 21. 14] were examined with various nucleus-substituted derivatives of Nα-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of α-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two subtilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar traps-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2, )-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5, 500±700s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.View full abstractDownload PDF (507K) -
Katsumi MATSUURA, Mitsuo NISHIMURA1978 Volume 84 Issue 3 Pages 539-546
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane potential jump was induced by the addition of valinomycin in the presence of a KCl concentration gradient across the membrane of Rhodopseudomonas sphaeroides chromatophores. As well as a carotenoid band shift, which is known to be an indicator of mem-brane potential, absorbance changes due to the oxidation-reduction reactions of cytochromes accompanied the jump. Under aerobic conditions with no reductant added, a part of cyto-chrome c2 was reduced by an inside-positive potential jump of about 100mV in the time range of tens of seconds. This can be explained by the location of the cytochrome on the inner side of the chromatophore membrane and electrophoretic flow of electrons across the membrane. On the other hand, in the presence of 1mM ascorbate, a similar jump of membrane potential induced a rapid oxidation of cytochrome c2 and a subsequent reduction. A rapid reduction of b-type cytochrome was also observed. Antimycin A inhibited the c2 oxidation, but did not inhibit the b reduction.
The oxidation of cytochrome c2 may be explained by a diffusion-potential-induced electron flow to cytochrome b and a simultaneous electron donation by cytochrome b and cytochrome c2 to a common electron acceptor, possibly a quinone.View full abstractDownload PDF (523K) -
Toshiaki IMAGAWA, Takao NAKAMURA1978 Volume 84 Issue 3 Pages 547-557
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane-bound NADH dehydrogenase, solubilized and partially purified from a marine bacterium Photobacterium phosphoreum, contains FAD as the prosthetic group, and is specific for NADH. Ferricyanide, various other redox dyes and cytochrome c can act as electron acceptors. The enzymatic activity when assayed with electron acceptors other than cytochrome c, is activated by monovalent cations (Na+ and K+) and deactivated by high concentrations of monovalent anions (SCN-, NO3-, and CI-) but not by phosphate ions. The enzymatic reaction follows a ping-pong mechanism and kinetic analysis of the enzyme showed that the activation by monovalent cations is due to increase of affinity of the enzyme for substrates; Vm was not affected. The increase of affinity was 62- and 46-fold for NADH and 57- and 31-fold for 2, 6-dichlorophenol indophenol in the presence of Na+ and K+, respectively. On the other hand, NADH-cytochrome c reductase activity of the enzyme was strongly inhibited by these cations.View full abstractDownload PDF (713K) -
Seiichi KOBAYASHI, Yutaka NAGAI1978 Volume 84 Issue 3 Pages 559-567
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree different types of neutral proteases related to collagen metabolism have been found in the granule fraction of human leucocytes from normal adults, using collagen, gelatin, and synthetic peptides as substrates. These are collagenase, an enzyme showing a potent hydrolytic activity against gelatin but little against native collagen, and one splitting the cross-links region of collagen. Their molecular weights were estimated to be about 75, 000 150, 000, and 25, 000, respectively, by gel chromatography.
The former two enzymes were inhibited by a α2-macroglobulin and ethylenediaminetetra-acetate, but not by α1-proteinase inhibitor (α1, - antitrypsin) or phenylmethylsulfonylfluoride, while the latter enzyme, associated in behavior with an enzyme hydrolyzing succinyl-(L-alanyl)3-p-nitroanilide, was inhibited by α1-proteinase inhibitor, α2-macroglobulin, and phenylmethyl-sulfonylfluoride, but not by ethylenediaminetetraacetate.View full abstractDownload PDF (3253K) -
Chemical and Catalytic PropertiesShinzaburo TAKAMIYA, Toshio FUKUI1978 Volume 84 Issue 3 Pages 569-574
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical and catalytic properties of potato phosphoglucomutase [EC 2. 7. 5. 1] were studied using various enzyme species (Peaks Ia, Ib, Ic, and II; Takamiya, S. & Fukui, T. (1978) Plant Cell Physiol. 19, 319-328). The molecular weights of the species are all approximately 60, 000. No indication of the presence of subunit structure was obtained under various conditions. The amino acid composition of Peak la is generally similar to those of the enzymes from other sources, though it has some peculiarities. The Peak Ia and Peak II enzymes both absolutely require α-D-glucose 1, 6-bisphosphate and Mg2+ for activity, and appear to have a “ping-pong” mechanism. A low concentration of Beet inhibits their action, the inhibition being retarded either by Mg2+ or EDTA. Although the inhibition patterns by various metabolites are similar for Peaks Ia and II, they differ in their kinetic parameters and optimal pH values.View full abstractDownload PDF (384K) -
Yasuo NAKANISHI, Masahiro TSUJI, Kazuhiko ISHIHARA, Shigemi KATO, Nobo ...1978 Volume 84 Issue 3 Pages 575-584
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe egg white of newly laid chicken egg was found to contain about 46μmol of UDP-N-acetylgalactosamine 4-sulfate, 34μmol of GDP-mannose, 6μmol of UDP-N-acetylhexosamine, and 1μmol of UDP-N-acetylgalactosamine 4, 6-bissulfate per liter. There was no significant difference between infertile and fertile eggs in the initial levels of the sugar nucleotides.
During incubation for 4 days, the nucleotide levels in infertile eggs showed little change while those in fertile eggs fell continuously until the complete disappearance of the nucleotides on the fourth day. Initial removal of the blastoderm from fertile eggs resulted in cessation of the reduction in nucleotide levels in the 2- to 4-day period after the operation.
The decrease of UDP-N-acetylgalactosamine 4-sulfate was followed by an increase of 1-phospho-N-acetylgalactosamine 4-sulfate then N-acetylgalactosamine 4-sulfate in the egg white. When UDP-N-acetylgalactosamine 4-[35S]sulfate or GDP-[14C]mannose was injected into the egg white of a fertile egg, the main feature of the metabolism of the labeled compounds was the successive hydrolysis of their pyrophosphate and phosphate bonds, with the formation of sugar 1-phosphate and sugar.
A significant activity of nucleotide pyrophosphatase was detected in the egg white in newly laid eggs (0-day egg). However, no such activity could be detected in egg-white specimens from 1-, 2-, and 3-day eggs. The results suggest that although the decrease of sugar nucleotides in the first day could be ascribed to the hydrolytic action of the enzyme originally present in the egg white, the decrease in the subsequent 3 days results from a more complex process in which the hydrolysis of the sugar nucleotides is related to the development of the embryo.View full abstractDownload PDF (814K) -
Chan F. LAM, Nancy SCHATZ1978 Volume 84 Issue 3 Pages 585-595
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA computer algorithm is presented to derive initial velocity rate equation for general enzyme raction mechanisms, including sequential as well as complicated random mechanisms. The method is based on the theory of graph and the theory of prime number. In complicated mechanisms, there are many pathways and hence many cycles. The values of the rate constants are constrained according to the principle of detailed balance: the product of rate constants in the clockwise direction of a cycle must equal to the product of the rate constants in the counter-clockwise direction of the same cycle. An algorithm is presented to derive the appropriate constraint equations. These constraint equations are arranged so that when the rate equation is used for estimating rate constants, the resulting rate constants would satisfy the principle of detailed balance automatically.View full abstractDownload PDF (697K) -
Eiji ITAGAKI, Masatoshi ARISAWA, Toichiro HOSOYA1978 Volume 84 Issue 3 Pages 597-606
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) of reduced porcine thyro-globulin revealed more than ten distinct bands with molecular weights ranging from 19, 000 to 330, 000. When the thyroglobulin preparation was previously dialyzed against distilled water for 3 days at 4°C, the bands with large molecular weights disappeared, leaving only several small-sized components. These submolecular protein components, designated as Sub. I, Sub. II, Sub. III, and Sub. IV, were isolated by gel filtration on a Sephadex G-200 column in 0.1% SDS-0.04M Tris acetate buffer (pH 7.4) at 40°C, and the preparations were confirmed by sedimentation equilibrium analysis to be nearly homogenous in size. The weight average molecular weights determined by the sedimentation equilibrium method were 17, 500, 37, 400, 50, 300, and 70, 800 for Sub. I, Sub. II, Sub. III, and Sub. IV, respectively. Similar values were obtained by other methods, gel filtration and SDS-gel electrophoresis. Thus, the relative size of these components was very close to 1:2:3:4. The contents of amino acids and carbohydrates of these components were very similar to each other and also to entire thyroglobulin. The contents of iodoamino acids were also not greatly different between them, except that thyroxine and triiodothyronine seemed to be significantly high in the smallest protein, Sub. I. Immunochemical study also revealed a strong resemblance between each of them and thyroglobulin. Based on these results, together with other works, the structure and function of thyroglobulin are discussed.View full abstractDownload PDF (2386K) -
III. Properties of Mg- and Ca ATPaseTakashi SHIMIZU1978 Volume 84 Issue 3 Pages 607-616
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of bovine platelet myosin were investigated with regards to its Mg- and Ca-ATPase [EC 3. 6. 1. 3] activities. The Mg-ATPase exhibited an initial burst of phosphate liberation, the size of which was slightly larger than 1 mol P1/480, 000g protein. The apparent Km value of the Mg-ATPase for ATP at a high ionic strength was very low (0.1μM). A Lineweaver-Burk plot of Ca-ATPase at a high ionic strength was almost linear and the allostericity suggested by Malik et al. ((1974) Biochem. Biophys. Res. Commun. 61, 1071) was not observed. The apparent Km value of Ca-ATPase for ATP was 10μM at a constant CaC12 concentration of 2mM or 54μM at a constant Ca:ATP ratio of 1:1. In the presence of 1mM EDTA at a high ionic strength, the apparent Km value for ATP was 0.25mM, and in the virtual absence of EDTA, 0.1mM.
At a high ionic strength, both Mg- and Ca-ATPase activities showed typical U-shaped pH dependences at 25°C or 0°C. While Mg-ATPase activity at a low ionic strength and 25°C showed a similar U-shaped dependence, the activity measured at 0°C exhibited a slightly different dependence in a neutral pH region. The difference suggested a difference in rate limiting steps of the enzyme reaction, which was clearly shown with a bend at 4-6°C in an Arrhenius plot of Mg-ATPase activity at a low ionic strength.
KCl concentration dependences of Mg- and Ca-ATPase activities were different from that of Ca-ATPase described previously and were similar to those for smooth muscle myosin; ATPase activities increased with increasing KCl concentration. The difference between previous and present results would be due to the difference in assay temperatures. The Mg-activated enzyme activity at a high ionic strength showed broad substrate specificity as de-scribed previously for Ca-activated enzyme activity. While urea brought about activation of both Mg- and Ca-ATPase activities by 50-80%, modification with NEM did not enhance Ca-ATPase activity. On the contrary, Mg-ATPase was activated by NEM by 56%.View full abstractDownload PDF (687K) -
Tetsunori TANAKA, Teruo NAKAJIMA1978 Volume 84 Issue 3 Pages 617-625
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-Aspartyl-methionine, -aspartic acid and -glutamic acid and γ-glutamyl-threonine and -glycine were isolated and identified in human urine by means of ion-exchange chromatography, highvoltage paper electrophoresis, acid hydrolysis and determination of N-terminal amino acids of the isolated compounds, and comparison of their behaviors in paper electrophoresis and chromatography with those of the authentic compounds.
The concentrations of acidic B-aspartyl dipeptides in human urine were determined using an amino acid analyzer. Their concentrations were as follows: β-aspartyl-glycine, male, 44.4±8.5, female, 61.4±18.9, child, 83.7±27.1; -alanine, male, 11.0±4.9, female, 20.7±12.0, child, 25.3±9.1; -glutamic acid, male, 10.0±3.7, female, 23.0±8.5, child, 20.4±7.5; -serine, male, 9.9±2.8, female, 13.6±3.8, child, 14.9±4.7; -aspartic acid, male, 4.3±1.0, female, 9.1±2.2, child, 18.4±6.5; -threonine, male, 3.9±0.9, female, 5.8±1.1, child, 13.2±4.9μmol/g creatinine (mean±S. D.). The order of the sum of their concentrations tended to be child>female>male.
Patients receiving intravenous hyperalimentation also excreted acidic β-aspartyl dipeptides into urine in amounts similar to those in females and in a pattern similar to that observed in healthy persons. This finding indicates that urinary β-aspartyl dipeptides were probably of endogenous origin because oral nutrition was stringently excluded in these patients.View full abstractDownload PDF (588K) -
Mamoru ISEMURA, Hiroko KOSAKA, Tokuji IKENAKA, Ryo KIDO, Toshiki YOSHI ...1978 Volume 84 Issue 3 Pages 627-632
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA fluorogenic substrate for α-L-iduronidase, 4-methylumbelliferyl α-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis.
Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low α-L-iduronidase activity.
The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.View full abstractDownload PDF (968K) -
Tetsuo MURO, Hiroshi NAKATANI, Keitaro HIROMI, Hidehiko KUMAGAI, Hidea ...1978 Volume 84 Issue 3 Pages 633-640
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding process of tyrosine phenol-lyase, a B6-enzyme, with inhibitors (L-alanine, n-alanine, and L-meta-tyrosine) or the substrate (L-tyrosine) was investigated kinetically by the stopped-flow method. Since a new absorption spectrum at around 500nm appears upon binding of the enzyme with the inhibitors or the substrate, the reactions were monitored by means of the visible spectral change. For the enzyme-inhibitor interaction, the concentration dependence of the reciprocal relaxation time observed by the stopped-flow method was analyzed and indicated a two-step mechanism, a fast bimolecular association followed by a slow unimolecular process,
E+I k1_??_k-1EI1k2_??_k-2 EI2
where EI1 and EI2, are structural isomers of the enzyme-inhibitor complexes and k1, k-1 etc., are rate constants. No visible spectral change was detected by the rapid-scan stopped-flow method in the fast association process between the enzyme and the inhibitors. In the case of L-alanine and L-meta-tyrosine, an additional very slow relaxational process was observed, preceded by the fast process. The reciprocal relaxation time was independent of the inhibitor concentration.
Upon binding of the enzyme with substrate, L-tyrosine, three separate relaxational processes with visible spectral changes were observed. By analysis of the concentration depend-ence of the reciprocal relaxation time of the fastest process, the association and dissociation rate constants were determined (k1=1.4×104M-1•s-1 and k-1=7.8 s-1). The concentration dependence of the reciprocal relaxation time for the middle process showed a saturation curve and the rate constants k2(=0.46s-1) and k-2(=0.15s-1) were determined assuming the above two-step mechanism. The value of k2 was nearly equal to the catalytic center activity, kcat (=0.50s-1), suggesting that the rate-determining step could be the first unimolecular process. The reciprocal relaxation time for the slowest relaxation process did not depend on the substrate concentration. The slowest process was assigned to the formation of an abortive intermediate branching from the main catalytic pathway.View full abstractDownload PDF (522K) -
Shohei MAEKAWA1978 Volume 84 Issue 3 Pages 641-646
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTetrahymena tubulin has a very low affinity for colchicine. The dissociation rate constant (k-1) and association rate constant(k+1) of Tetrahymena tubulin for colchicine were determined in comparison with those of porcine brain tubulin.
The k+1 value of Tetrahymena tubulin was about one-twentieth that of porcine brain tubulin, and k-1 of Tetrahymena tubulin was about 680 times greater than that of brain tubulin. The dissociation constant(k-1/k+1) of Tetrahymena tubulin (2.7×10-3) turned out to be ten thousand times greater than that of brain tubulin.
This large Kd value explains well the high colchicine concentration requirement reported to block cell division or cilia regeneration of this protozoan.View full abstractDownload PDF (376K) -
Isamu SHIIO, Kyoko UJIGAWA1978 Volume 84 Issue 3 Pages 647-657
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlutamate-auxotrophic mutants lacking phosphoenolpyruvate carboxylase(PC), citrate synthase (CS) or glutamate dehydrogenase (GD), an aspartate auxotroph lacking aspartate aminotransferase (TA), and a glutamate-aspartate double auxotroph lacking both aconitase (AH) and TA were obtained from Brevibacterium flavum No. 2247, a glutamate-producing bacterium. Prototrophic revertants further derived from the CS- and GD-lacking auxotrophs concomitantly recovered the enzyme activities that their parents had lost. These results indicate involvement of the tricarboxylic acid (TCA) cycle and GD in glutamate biosynthesis, that of PC in the biosynthesis of the TCA cycle intermediates and that of TA in aspartate biosynthesis. The CS-deficient mutants accumulated large amounts of acetate and small amounts of pyruvate, aspartate and alanine, while the GD-deficient strains accumulated large amounts of 2-oxo-glutarate and small amounts of citrate.
Synthesis of PC was repressed by either glutamate or aspartate and those of CS and GD were repressed by glutamate, whereas those of pyruvate dehydrogenase (PD), AH, and isocitrate dehydrogenase were not affected significantly by glutamate; that of TA was also not affected by aspartate or by glutamate. The specific activities of PD and AH gave peaks during the cellular cultivation, related to the temporary accumulation of their substrates, pyruvate and citrate, respectively.
These and previous results on the regulation of the enzymatic activities provide a definite regulatory mechanism for glutamate and aspartate syntheses.View full abstractDownload PDF (787K) -
Takae TOWATARI, Kazuyoshi TANAKA, Daisuke YOSHIKAWA, Nobuhiko KATUNUMA1978 Volume 84 Issue 3 Pages 659-671
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1], was purified about 2, 200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies.
2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate.
3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22, 000, which is smaller than that of cathepsin B.
4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0-5.5. The enzyme was unstable in alkali and on heat treatment.
5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.View full abstractDownload PDF (1307K) -
Isamu YAMAMOTO, Makoto ISHIMOTO1978 Volume 84 Issue 3 Pages 673-679
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSE. coli K10 was found to grow anaerobically on molecular hydrogen by reducing nitrate, fumarate, and trimethylamine N-oxide when peptone was added to the culture medium. Molar growth yields based on consumed hydrogen estimated from the amounts of reduction products were all 7.8g cells/mol, suggesting that 1mol of ATP was produced in the oxidation of 1mol of hydrogen. Hydrogenase activity measured in terms of hydrogen evolution was several times higher in cells grown on glucose than in cells grown on hydrogen in the presence of fumarate and trimethylamine N-oxide, while hydrogenase activity measured in terms of hydrogen uptake was unchanged in both cases. The ratio of hydrogenase activities measured in terms of hydrogen uptake and evolution was also high in the extract and centrifugal fractions from cells grown on hydrogen. The soluble fraction and trypsin digest of the precipitate at 100, 000×g were subjected to polyacrylamide disc gel electrophoresis and hydrogenase bands were stained by reduction of benzyl viologen with hydrogen and by oxidation of reduced methyl viologen. The resulting patterns suggest that multiple forms of hydrogenase are present and that the amounts of forms functioning in hydrogen evolution were greatly decreased in cells grown on hydrogen in the presence of acceptors.View full abstractDownload PDF (1032K) -
Hiroshi MORI, Yasuo FUKAMI, Ichiro HARUNA1978 Volume 84 Issue 3 Pages 681-686
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhage SP RNA-dependent RNA polymerase (SP replicase) was purified from Escherichia coli infected with RNA phage SP. The enzyme was found to be composed of four non-identical polypeptides, i.e. subunits I, II, III, and IV with molecular weights of 74, 000, 69, 000, 47, 000, and 36, 000 daltons, respectively. As in the case of phage Qβ replicase, the largest polypeptide is identical with the ribosomal protein SI, and subunits III and IV with polypeptide chain elongation factors EF-Tu and EF-Ts, respectively. This is based on the behaviour of the subunits on SDS-polyacrylamide gel electrophoresis, isoelectric focusing and immunological cross-reaction. Subunits I, III, and IV of SP replicase are derived from the host cell, while subunit II is coded by phage RNA genome.
The striking coincidence of the composition and entity of the structural components of SP replicase with those of Qβ replicase may indicate the structural and functional requirements of host-derived polypeptides in RNA replicase.
The binding activity of Sl (in 70S ribosome complex) to poly (U) is retained in SP replicase complex. In contrast, the GDP binding activity of EF-Tu is masked in SP replicase. It is concluded that S1 is required functionally whereas EF-Tu•EF-Ts are required structurally in RNA replicase.View full abstractDownload PDF (1508K) -
I. The Incorporation of Radioactivity into NADTakashi UEDA, Hidetsugu OTSUKA, Kiyoshi GODA, Isao ISHIGURO, Junko NAI ...1978 Volume 84 Issue 3 Pages 687-696
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1×8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.View full abstractDownload PDF (706K) -
II. Substrate Specificity of Microsomal Deacetylation of TrichothecenesMinoru OHTA, Hiroko MATSUMOTO, Kenji ISHII, Yoshio UENO1978 Volume 84 Issue 3 Pages 697-706
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe substrate specificity of microsomal nonspecific carboxyesterase [EC 3.1.1.1] from rabbit and rat livers was studied in vitro by using seven (A)-type and six (B)-type 12, 13-epoxytrichothecene mycotoxins. The C-4 acetyl residues of diacetoxyscirpenol, T-2 toxin, monoacetylnivalenol (fusarenon-X), and diacetylnivalenol were selectively hydrolyzed by the microsomal esterase to yield the corresponding C-4-deacetylated metabolites: monoacetoxyscirpenol, HT-2 toxin, nivalenol, and 15-acetylnivalenol, respectively. The C-3 acetyl group of monoacetyldeoxy-nivalenol and the C-8 acetyl group of tetraacetoxyscirpen were also deacetylated. Triacetoxyscirpen gave rise to two unidentified metabolites, which may include a C-4-deacetylated product. 8-Hydroxydiacetoxyscirpenol (neosolaniol), HT-2 toxin, acetyl-T-2 toxin and tetraacetylnivalenol were unaffected by this type of hydrolysis. It follows from these results that the C-4 acetyl residue is hydrolyzed by the microsomal carboxyesterase and substituents at C-3 and C-8 contribute to the selective enzymatic hydrolysis of the C-4 acetyl residue of trichothecenes. Kinetic analysis showed that rabbit microsomal esterase possessed a high affinity for (A)-type trichothecenes such as T-2 toxin and diacetoxyscirpenol, and that of rat microsomes possessed a high affinity for (B)-type trichothecenes such as monoacetylnivalenol (fusarenon-X).
The significance of this specific deacetylation reaction is discussed in relation to the bio-logical activity of the trichothecene derivatives as revealed by their inhibitory effect on protein synthesis in rabbit reticulocytes.View full abstractDownload PDF (678K) -
Hideyo HASUMI, Satoshi NAKAMURA1978 Volume 84 Issue 3 Pages 707-717
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular weight of spinach ferredoxin-NADP reductase [EC 1.6.99.4] was estimated to be 33, 100 by the sedimentation equilibrium method. On the basis of this molecular weight, the amino acid composition of the reductase was determined.
The reactivity of ferredoxin toward p-chloromercuribenzoate was investigated. By measuring the time course of the reaction, 1mol of ferredoxin was found to react with about 8mol of p-chloromercuribenzoate in 10min. Under low ionic strength conditions (1mM NaCl), the second-order rate constants of this reaction determined spectrophotometrically at 420 and 250mn were 3, 640 and 3, 690M-1•S-1, respectively; under high ionic strength conditions (100mM NaCl), these rate constants were 1, 360 and 1, 270M-1•-S-1, respectively. In the presence of the reductase, the rate constants under low and high ionic strength condi-tions were 54 and 1, 040M-1•S-1, respectively.
By investigation of the solvent perturbation effects on the aromatic amino acid residues with 20% ethylene glycol, it was found that ferredoxin, ferredoxin-NADP reductase, and the complex between these proteins had 2.8, 6.3, and 3.8mol of exposed tyrosyl residues per mol of protein, respectively. It therefore seems likely that about 5 tyrosyl residues may exist in the neighborhood of the binding site of the complex of these proteins.View full abstractDownload PDF (717K) -
Hideo INOUE, Akira ASADA, Yukio KATO, Yoshiro TAKEDA1978 Volume 84 Issue 3 Pages 719-725
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe metabolism of spermidine, and especially putrescine formation from spermidine during the prereplicative period of cell proliferation, was investigated in isoproterenol-stimulated mouse parotid glands.
The rate of spermidine synthesis from putrescine decreased significantly about 4h after isoproterenol treatment, whereas the putrescine level increased at this time. Spermidine synthesis began to increase from 6h after isoproterenol treatment, and was maximal 8 to 10h after treatment.
Administration of isoproterenol resulted in a gradual decrease in the spermidine con-centration to a minimum of about 80%. of the control level after 6h. This decrease was accompanied by increased conversion of spermidine to putrescine. The degradation of spermidine to putrescine contributed to the increase in the putrescine level in the parotid glands in the very early stage after isoproterenol treatment, the contribution reaching more than 70% at 4 and 6h after treatment. No increased degradation of spermidine to putrescine was observed in the liver or kidney, where cell proliferation was not accelerated by isoproterenol.
Administration of isoproterenol had little effect on conversion of spermidine to spermine or the reverse reaction in parotid glands.
These results show that the increase in the putrescine level observed in mouse parotid glands soon after isoproterenol treatment is due to not only an increase in ornithine decarboxylase activity but also enhanced conversion of spermidine to putrescine. The results also suggest that putrescine is not merely used for spermidine synthesis in the early prereplicative stage, and that the two amines may be of different importance for growth in this system.View full abstractDownload PDF (550K) -
Akira OKURA, Shonen YOSHIDA1978 Volume 84 Issue 3 Pages 727-732
Published: September 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of 9-β-D-arabinofranosyladenine-5'-triphosphate (araATP) on the reactions of DNA polymerases α and β [E.C. 2.7.7.7] purified from calf thymus was examined. The reaction of DNA polymerase α was shown to be more sensitive to the inhibition than that of DNA polymerase β. The K1 value of DNA polymerase β for araATP was 45μm; 15times higher than that of DNA polymerase α (3μM). The mode of inhibition by araATP was essentially competitive to deoxyadenosine triphosphate (dATP) in the reactions catalyzed by both DNA polymerase α and β using activated DNA as a template-primer. However, in the reactions of the α-enzyme, araATP also inhibited the incorporation of deoxyribonucleotides other than dATP non-competitively.View full abstractDownload PDF (442K) -
1978 Volume 84 Issue 3 Pages 733a
Published: 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (18K) -
1978 Volume 84 Issue 3 Pages 733b
Published: 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (18K)
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